Pneumocystis carinii is an opportunistic fungal pathogen which causes severe pneumonia in patients with an impaired immune system, particularly in patients with AIDS. The life cycle of P. carinii is poorly understood. In closely related fungi, Cdc2 associated with a regulatory cyclin controls the transition through the G1 restriction point START in the cell cycle.1-7 In fungi homologous to P. carinii, pheromone mating factors inhibit START through a mitogen-activated protein kinase (MAPK) signal transduction cascade which results in cell cycle arrest and cellular differentiation.8-14 MAPK phosphorylates an inhibitor of the Cdc2-cyclin complex.15-18 MAPK is the essential molecule in the pheromone mating cascade which controls the transition from mitotic growth to a pathway of conjugation, cellular differentiation, and proliferation.8 As an essential molecule regulating an organism's life cycle, MAPK is regulated by a kinase cascade and is activated by phosphorylation on two conserved threonine and tyrosine amino acids.19-24 Attachment of P. carinii to the alveolar epithelium is a central feature of P. carinii pneumonia, and binding to the epithelium is required for the organisms to proliferate.25-26 Our initial studies indicate that P. carinii possesses a MAPK which is highly homologous to fungal MAPK's involved in mating.27 We, therefore, hypothesize that the MAPK signal transduction cascade has a central role in the regulation of the P. carinii life cycle. We further propose that the P. carinii MAPK regulates cellular differentiation and proliferation of P. carinii organisms. To evaluate these hypotheses, we will undertake several parallel investigations. We will characterize the P. carinii MAPK genes involved in life cycle regulation. We will determine the kinase activity and the regulatory phosphorylations for P. carinii MAPK. Further, we will determine whether the expression and activity of P. carinii MAPK is differentially regulated throughout the life cycle of P. carinii by evaluating MAPK in separated cysts and trophozoites. Finally, we will determine the role of MAPK in the transition from one life form to another by evaluating the expression and activity of P. carinii MAPK before and after attachment to the alveolar epithelium and by overexpressing MAPK genes in separated life cycle forms of P. carinii. Through these investigations, we hope to gain important insights into the life cycle regulation of P. carinii.